Burkholderia pseudomailei is the causative agent of melioidosis, a global emerging infectious disease, and it is also classified as a category B potential bioterrorism agent. Thousands of cases of melioidosis have occurred in the tropics, and several have been reported in American travelers (civilian and military personnel). Other cases have been documented in Hawaii, and the bacterium has been detected on the Big Island of Hawaii. Using a novel method we recently pioneered to profile global gene-expression in single prokaryotic cells, we analyze the 'transitome' of S. pseudomailei as the bacterium transits through the hostcell during its intracellular infectious lifecycle. Our preliminary data revealed expression of a series of hypothetical proteins and transcriptional regulators in unique infectious stages of B. pseudomailei. Highthrough-put chromosomal mutagenesis of 200 unique hypothetical-protein-encoding genes, expressed in a stage-specific fashion, and initial phenotype screening narrowed the list to 11 hypothetical proteins that decreased virulence and the ability of 6. pseudomailei to infect host-cells. This application proposes to continue this research through two aims: i) To firmly validate the transitomic data by independent methods using reporter-gene-fusion and single-cell real-time RT-PCR and continue with identifying the importance of the 11 hypothetical proteins using host-cell infection models and a murine melioidosis model of infection, and ii) To generate 45 unique regulatory mutants on the B. pseudomailei chromosome to identify their importance in cell and murine melioidosis models. These studies will yield further insight into the functions of unknown virulence genes expressed in a stage-specific fashion in the 6. pseudoma//e/transitome and help elucidate the molecular mechanisms involved in B. pseudomailei intracellular infection.